Kim Youngmi, Phillips Joseph A, Liu Haipeng, Kang Huaizhi, Tan Weihong
Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA.
Proc Natl Acad Sci U S A. 2009 Apr 21;106(16):6489-94. doi: 10.1073/pnas.0812402106. Epub 2009 Apr 9.
The ability to inhibit an enzyme in a specific tissue with high spatial resolution combined with a readily available antidote should find many biomedical applications. We have accomplished this by taking advantage of the cis-trans photoisomerization of azobenzene molecules. Specifically, we positioned azobenzene moieties within the DNA sequence complementary to a 15-base-long thrombin aptamer and then linked the azobenzene-modified cDNA to the aptamer by a polyethylene glycol (PEG) linker to make a unimolecular conjugate. During the photoisomerization of azobenzene by visible light, the inhibition of thrombin is disabled because the probe hybridizes with the cDNA in the trans-azobenzene conformation so that the aptamer cannot bind its target thrombin. However, when UV light is applied, melting of the hairpin structure (duplex) is induced via trans-to-cis conversion, thereby changing conformation of the aptamer and making the aptamer free to bind to and inhibit its target thrombin. By using standard clotting assays, we measured the IC(200) of various probe designs in both states and concluded the feasibility of using photon energy to temporally and spatially regulate these enzymatic reactions. Thus, we can report the development of DNA probes in the form of photon-controllable (thrombin) inhibitors, termed PCIs, and we expect that this approach will be highly beneficial in future biomedical and pharmaceutical applications.
能够以高空间分辨率在特定组织中抑制一种酶，并结合易于获得的解毒剂，这将在许多生物医学应用中找到用武之地。我们通过利用偶氮苯分子的顺反光异构化实现了这一点。具体而言，我们将偶氮苯部分定位在与15个碱基长的凝血酶适配体互补的DNA序列中，然后通过聚乙二醇（PEG）连接子将偶氮苯修饰的cDNA与适配体连接起来，形成单分子共轭物。在可见光使偶氮苯发生光异构化过程中，凝血酶的抑制作用被解除，因为探针以反式偶氮苯构象与cDNA杂交，使得适配体无法结合其靶标凝血酶。然而，当施加紫外光时，通过反式到顺式的转变诱导发夹结构（双链体）解链，从而改变适配体的构象，使适配体能够自由结合并抑制其靶标凝血酶。通过使用标准凝血测定法，我们测量了两种状态下各种探针设计的IC(200)，并得出利用光子能量在时间和空间上调节这些酶促反应的可行性。因此，我们可以报道以光子可控（凝血酶）抑制剂形式存在的DNA探针的开发，称为PCI，并且我们预计这种方法在未来的生物医学和制药应用中将非常有益。
